Excitatory action of γ-aminobutyric acid (GABA) on crustacean neurosecretory cells

Summary 1. Intracellular and voltage-clamp recordings were obtained from a selected population of neuroscretory (ns) cells in the X organ of the crayfish isolated eyestalk. Pulses of -aminobutyric acid (GABA) elicited depolarizing responses and bursts of action potentials in a dose-dependent manner. These effects were blocked by picrotoxin (50 µM) but not by bicuculline. Picrotoxin also suppressed spontaneous synaptic activity.2. The responses to GABA were abolished by severing the neurite of X organ cells, at about 150 µm from the cell body. Responses were larger when the application was made at the neuropil level.3. Topical application of Cd²⁺ (2 mM), while suppressing synaptic activity, was incapable of affecting the responses to GABA.4. Under whole-cell voltage-clamp, GABA elicited an inward current with a reversal potential dependent on the chloride equilibrium potential. The GABA effect was accompanied by an input resistance reduction up to 33% at a –50 mV holding potential. No effect of GABA was detected on potassium, calcium, and sodium currents present in X organ cells.5. The effect of GABA on steady-state currents was dependent on the intracellular calcium concentration. At 10^–6 M [Ca²⁺]_i, GABA (50 µM) increased the membrane conductance more than threefold and shifted the zero-current potential from–25 to–10 mV. At 10^–9 M [Ca²⁺]_i, GABA induced only a 1.3-fold increase in membrane conductance, without shifting the zero-current potential.6. These results support the notion that in the population of X organ cells sampled in this study, GABA acts as an excitatory neurotransmitter, opening chloride channels.     

Role of uptake inγ-aminobutyric acid (GABA)-mediated responses in guinea pig hippocampal neurons

Intracellular recordings were obtained from hippocampal pyramidal neurons maintained in vitro. Measurements were made of the conductance change induced by iontophoretically applied gamma-aminobutyric acid (GABA) and, using voltage-clamp techniques, of inhibitory postsynaptic currents resulting from activation of inhibitory pathways. Analysis of GABA iontophoretic charge-response curves indicated that there was considerable variation among neurons with respect to the slope of this relation. The placement of the GABA-containing pipette did not appear to be responsible for the observed variation, since vertical repositioning of the pipette did not alter the slope of the charge-response relationship. Steady iontophoresis of GABA from one barrel of a double-barreled pipette markedly affected the charge-response relation obtained when short pulses were applied to the other barrel. The curve was shifted to the left, and the slope was decreased. Concomitantly, the enhanced GABA-induced responses were prolonged. Similar alterations in GABA responsiveness were observed when the uptake blocker, nipecotic acid, was iontophoretically applied. Furthermore, bath application of saline containing a reduced sodium concentration (25% of control) also produced a prolongation of GABA-mediated responses. Under voltage clamp, inhibitory postsynaptic currents were observed to have biphasic decays. The initial, fast decay was prolonged by an average of 18% by nipecotic acid, whereas the later, slow phase was prolonged by 23%. The results of these studies support the hypothesis that a saturable GABA uptake system is responsible for the observed variation in the charge-response curves and, in turn, underlies the apparent sensitizing effect of excess GABA application. The results also suggest that a reduction of transmitter uptake affects the time course of inhibitory postsynaptic currents in the hippocampus.     

Cellular distribution ofl-glutamate decarboxylase (GAD) andγ-aminobutyric acidA (GABAA) receptor mRNAs in the retina

1. Gamma-aminobutryic acid (GABA), a major inhibitory transmitter of the vertebrate retina, is synthesized from glutamate by L-glutamate decarboxylase (GAD) and mediates neuronal inhibition at GABAA receptors. GAD consists of two distinct molecular forms, GAD65 and GAD67, which have similar distribution patterns in the nervous system (Feldblum et al., 1990; Erlander and Tobin, 1991). GABAA receptors are composed of several distinct polypeptide subunits, of which the GABAA alpha 1 variant has a particularly extensive and widespread distribution in the nervous system. The aim of this study was to determine the cellular localization patterns of GAD and GABAA alpha 1 receptor mRNAs to define GABA- and GABAA receptor-synthesizing neurons in the rat retina. 2. GAD and GABAA alpha 1 mRNAs were localized in retinal neurons by in situ hybridization histochemistry with 35S-labeled antisense RNA probes complementary to GAD67 and GABAA alpha 1 mRNAs. 3. The majority of neurons expressing GAD67 mRNA is located in the proximal inner nuclear layer (INL) and ganglion cell layer (GCL). Occasional GAD67 mRNA-containing neurons are present in the inner plexiform layer. Labeled neurons are not found in the distal INL or in the outer nuclear layer (ONL). 4. GABAA alpha 1 mRNA is expressed by neurons distributed to all regions of the INL. Some discretely labeled cells are present in the GCL. Labeled cells are not observed in the ONL. 5. The distribution of GAD67 mRNA demonstrates that numerous amacrine cells (conventional, interstitial, and displaced) and perhaps interplexiform cells synthesize GABA. These cells are likely to employ GABA as a neurotransmitter. 6. The distribution of GABAA alpha 1 mRNA indicates that bipolar, amacrine, and perhaps ganglion cells express GABAA receptors having an alpha 1 polypeptide subunit, suggesting that GABA acts directly upon these cells.     

Regulation of potassium levels by Müller cells in the vertebrate retina

The membrane properties of Müller cells, the principal glial cells of the vertebrate retina, have been characterized in a series of physiological experiments on freshly dissociated cells. In species lacking a retinal circulation (tiger salamander, rabbit, guinea pig), the end-foot of the Müller cell has a much higher K+ conductance than do other cell regions. In species with retinal circulation (mouse, cat, owl monkey) the K+ conductance of the end-foot is greater than the conductance of the proximal process of the cell. In these species, however, the K+ conductance of the soma and distal process is equal to, or greater than, the end-foot conductance. Müller cells also possess four voltage-dependent ion channels, including an inward rectifying K+ channel. These membrane specializations may aid in the regulation of extracellular K+ levels by Müller cells in the retina. High end-foot conductance shunts excess K+ out through the end-foot, where it diffuses into the vitreous humor. In vascularized retinae, excess K+ may also be transferred to the ablumenal wall of capillaries, where it could be transported into the blood.     

Autoradiographic studies of the γ-aminobutyric acid (GABA) system in the rat pancreas

Summary The -cells of the pancreatic islets have been shown to contain -aminobutyric acid (GABA) together with insulin. Autoradiographic analysis indicated that high affinity GABA binding sites (GABA receptors) are not present in the pancreas. High affinity GABA uptake sites are present, not in -cells, but in a few cells on the periphery of the islets. These observations cast doubt on the suggestion that GABA has a paracrine role in the pancreas.     

Inhibition of transporter mediated γ-aminobutyric acid (GABA) release by SKF 89976-A,a GABA uptake inhibitor,studied in a primary neuronal culture from chicken

The effect of SKF 89976-A, a lipophilic non-substrate inhibitor of the -aminobutyric acid (GABA) transporter, on the release of radioactive GABA andd-aspartate has been studied. Neuronal cultures from 8 day old chick embryos, grown for six days, served as a model. The cultures were incubated with [³H]d-aspartate and [¹⁴C] GABA with the subsequent addition of high or low concentrations of SKF 89976-A. Finally the cultures were exposed to differently composed media for either 30 or 300 seconds. The release was quantified, using liquid scintillation counting. The efflux of [³H]d-aspartate and [¹⁴C] GABA was increased by [K⁺] and time, and a minimum value was obtained at [Ca²⁺] 1.05 mM. The release of both [³H]d-aspartate and [¹⁴C] GABA was inhibited by SKF 89976-A. The obtained results indicate that transporter mediated processes are the major mechanisms of transmitter release in the investigated model.     

Glutamic Acid Independent Production of Poly(γ-glutamic acid) by Bacillus subtilis TAM-4

A bacterium that produced a large amount of poly(γ-glutamic acid) (PGA) when it was grown aerobically in a culture medium containing ammonium salt and sugar as sources of nitrogen and carbon, respectively, was isolated from soil. The bacterium, strain TAM-4, was classified as Bacillus subtilis. The maximum PGA production (22.1 mg/ml) was obtained when it was grown in a medium containing 1.8% ammonium chloride and 7.5% fructose at 30°C for 96 h with shaking. Some properties of the PGA obtained at different times of cultivation were investigated by gel permeation chromatography, SDS–PAGE, and measurement of viscosity, and calculation of the d/l ratio of glutamic acid constituting PGA. The results suggested that PGA was elongated with no changes in the diastereoisomer ratio in the molecule.     

Regulation of γ-aminobutyric acid synthesis in the vertebrate nervous system

The regulation of glutamic decarboxylase (GAD) activity is undoubtedly the key to the control of the steady-state concentrations of 4-aminobutyric acid (GABA) in the central nervous system. Those factors that might influence GAD activity are reviewed. They include repression and induction of GAD synthesis; the interconversion of the holo- and apo-form of GAD; the availability of substrate and cofactor; the competitive inhibition of GAD by endogenous substances, including GABA; and the involvement of calcium ions in whole-cell preparations. Where possible mechanisms of action are described, and the likelihood that each is of physiological importance is discussed. Experiments are suggested that would help clarify (1) the role of GABA in GAD repression; (2) the possible phosphorylation of GAD; and (3) the existence of multiple forms of the enzyme. In addition, a kinetic mechanism is proposed to explain the possible regulation of GAD by the interconversion of the holo- and apo-forms of the enzyme. It is concluded that the overriding factors responsible for GAD regulation are not yet understood. However, a possible mechanism relying on the direct feedback action of GABA on GAD activity has many attractive features.     

Interactions ofγ-aminobutyric acid (GABA), pentobarbital,and homopantothenic acid (HOPA) on internally perfused frog sensory neurons

Augmentatory actions among Cl- currents (ICl) induced by gamma-aminobutyric acid (GABA), pentobarbital (PB), and homopantothenic acid (HOPA) were investigated in isolated frog sensory neurons after suppression of Na+, K+, and Ca2+ currents using a suction pipette technique which combines internal perfusion with voltage clamp. GABA-sensitive neurons responded to both PB and HOPA, and the responses behaved as a simple Cl- electrode and reversed at the Cl- equilibrium potential (ECl). The dose-response curve for GABA-induced Cl- conductance was sigmoidal with the GABA concentration producing a half-maximum response (4.2 X 10(-5) M). Both GABA and HOPA dose-response curves shifted to the left in the presence of PB, though the facilitatory action of PB on GABA- and HOPA-induced ICl was more effective in the former. There was a significant facilitatory interaction between GABA- and HOPA-induced ICl. It is concluded that HOPA affects the GABA-GABA or PB-PB receptor interactions.     

Damage to the high-affinity γ-aminobutyric acid (GABA) uptake system in mouse brain by horseradish peroxidase (HRP)

Presynaptic nerve terminals when depolarized are sensitive to morphological and functional alteration by horseradish peroxidase. Mouse brain slices, 0.1 mm, depolarized by a K⁺-HEPES buffer and exposed to horseradish peroxidase exhibited alterations in both synaptic vesicle membrane structure and in high-affinity [¹⁴C]γ-aminobutyric acid uptake. The post stimulatory retrieval of synaptic vesicles from the nerve terminal plasma membrane in the presence of horseradish peroxidase resulted in a decrease in the synaptic vesicle population with a concurrent increase in non-synaptic vesicle membrane structures. High-affinity [¹⁴C]γ-aminobutyric acid uptake into 0.1-mm slices of mouse cerebral cortex and ponsmedulla-spinal cord was inhibited by 31% and 24%, respectively, after incubation for 60 min in K⁺-HEPES buffer containing horseradish peroxidase. Superoxide dismutase protected both the synaptic vesicle membrane and the high-affinity uptake system from the deleterious effects of horseradish peroxidase, pointing to the possible involvement of superoxide anion radicals in the horseradish peroxidase-related effects. These horseradish peroxidase induced alterations appear to be directed towards the exposed synaptic vesicle membrane, since non-stimulated brain slices exposed to horseradish peroxidase do not exhibit a reduction in either high- or low-affinity [¹⁴C]γ-aminobutyric acid uptake. Low-affinity uptake of [¹⁴C]γ-aminobutyric acid and [¹⁴C]α-aminoisobutyric acid into cortical slices was not affected after incubation in K⁺-HEPES with horseradish peroxidase. Low-affinity uptake, however, is reduced by the high-K⁺/Na⁺-free stimulatory incubation prior to uptake. It appears, thus, that high- and low-affinity uptake are distinct and different systems, with the high-affinity transport system structurally associated with synaptic vesicle membrane.     

Release of ATP from avian Müller glia cells in culture

ATP can be released from neurons and act as a neuromodulator in the nervous system. Besides neurons, cortical astrocytes also are capable of releasing ATP from acidic vesicles in a Ca(2+)-dependent way. In the present work, we investigated the release of ATP from Müller glia cells of the chick embryo retina by examining quinacrine staining and by measuring the extracellular levels of ATP in purified Müller glia cultures. Our data revealed that glial cells could be labeled with quinacrine, a reaction that was prevented by incubation of the cells with 1μM bafilomycin A1 or 2μM Evans blue, potent inhibitors of vacuolar ATPases and of the vesicular nucleotide transporter, respectively. Either 50mM KCl or 1mM glutamate was able to decrease quinacrine staining of the cells, as well as to increase the levels of ATP in the extracellular medium by 77% and 89.5%, respectively, after a 5min incubation of the cells. Glutamate-induced rise in extracellular ATP could be mimicked by 100μM kainate (81.5%) but not by 100μM NMDA in medium without MgCl(2) but with 2mM glycine. However, both glutamate- and kainate-induced increase in extracellular ATP levels were blocked by 50μM of the glutamatergic antagonists DNQX and MK-801, suggesting the involvement of both NMDA and non-NMDA receptors. Extracellular ATP accumulation induced by glutamate was also blocked by incubation of the cells with 30μM BAPTA-AM or 1μM bafilomycin A1. These results suggest that glutamate, through activation of both NMDA and non-NMDA receptors, induces the release of ATP from retinal Müller cells through a calcium-dependent exocytotic mechanism.     

Decreasedγ-aminobutyric acid (GABA) modulatory effect on rat vas deferens neurotransmission after chronic administration of imipramine

1. Chronic (10 mg/kg, i.p., once daily for 14 days) but not acute (10 mg/kg, i.p., 24 hr) administration of imipramine resulted in a decrease in both the responsiveness and the sensitivity of the contractions of the isolated rat vas deferens elicited by field stimulation to GABA and (-)-baclofen. 2. In contrast, clonidine and isoproterenol effects were not altered by either treatment. 3. This study shows for the first time that GABA action in the peripheral nervous system is altered by chronic treatment with antidepressants, possibly by inducing changes in a postreceptor element.     

Decreased emodepside sensitivity in unc-49 γ-aminobutyric acid (GABA)-receptor-deficient Caenorhabditis elegans

Emodepside, a semi-synthetic derivative of PF1022A, belongs to a new class of anthelmintic drugs, the cyclooctadepsipeptides, and shows good efficacy against macrocyclic lactone-, levamisole- or benzimidazole-resistant nematode populations. Although putative receptors for emodepside have already been discovered, its mode of action is still not fully understood. The involvement of the γ-aminobutyric acid (GABA)-receptor on the PF1022A mode of action has previously been postulated. Therefore, a possible role of the GABA-receptor, unc-49, in the mode of action of emodepside was investigated using two different Caenorhabditis elegans in vitro assays, a motility assay and a development assay. It was found that there is a clearly reduced sensitivity against emodepside of strains carrying a GABA-receptor, unc-49, loss of function mutation compared with N2 wild type C. elegans. To transfer these results from the model system to parasitic nematodes, the Toxocara canis unc-49B cDNA sequence was identified and used in a rescue experiment. The emodepside-susceptible phenotype could be fully rescued by injection of the T. canis unc-49B cDNA sequence. We believe that this is the first functional rescue of a C. elegans mutant strain with a gene from a clade III parasitic nematode. These findings, together with the earlier data on GABA-receptor binding of PF1022A, suggest that the GABA(A)-receptor UNC-49 is associated with the emodepside mode of action. However, the only partially resistant phenotype of the loss of function mutants indicates that other pathways play a more significant role.     

Regulation of swelling—activated chloride channels in embryonic chick heart cells

Swelling-activated Cl^- currents,I(Cl,swell),were measured during hyposmotic shock in white Leghorn embryonic chick heart cells using the whole-cell recording of patch-clamp technique.Genistein,an inhibitor of protein tyrosine kinase(PTK),suppressed I(Cl,swell).Under isosmotic condition phorbol 12-myristate 13-actetate(PMA),and activator of PKC,elicited the Cl^- current similar to that in hyposmotic solution,whereas hyposmotic shock did not elicit I(Cl,swell) in chelerythrine chloride(an inhibitor of PKC)-treated cells,Confocal microscopy experiments using FITC-phalloidin as a fluorescent label of F-actin showed that the actin network was moved from cortical region of the cell to the center after hyposmotic shock as compared with the image under isosmotic condition,When the cells were treated with cytochalasin B(CB)or cytochalasin D(CD)under isosmotic condition the disruption of the F-actin integrity was observed,and I(C,l,swell). The results suggested that the role of PTK,probably receptor tyrosine kinase,for regulation of I(Cl,swell) appeared to be at upstream site related to the role of F-actin.Then PKC signal pathway was activated somehow and finally change in the polymerization state of cytoskeleton led to activate the swelling-activated Cl^- channels.These results demonstrate clearly that PTK,PKC and F-actin are important factors for regulation of I(Cl,swell),in embryonic chick heart cells as compared with often controversial results reported in different cell types.     

Cloning of a putative γ-aminobutyric acid (GABA) receptor subunit ϱ3 cDNA

Cloned cDNA encoding a putative member of GABA receptor ϱ-subunit class was isolated from rat-retina-mRNA-derived libraries. The cDNA encodes a signal peptide of 21 amino acids followed by the mature ϱ3 subunit sequence of 443 amino acids. The proposed amino acid sequence exhibits 63 and 61% homology to the previously-reported human ϱ1 and rat ϱ2 sequences, respectively. Northern blot analysis demonstrated the expression of mRNA for ϱ3 subunit in retina.